Besides the four major nucleosides (2′-deoxycytidine (dC), 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG) and 2′-deoxythymidine (dT)), DNA of most living organisms contains minor amounts of methylated nucleosides: 5-methyl-2′-deoxycytidine (dCmethyl), N4-methyl-2′-deoxycytidine and N6-methyl-2′-deoxyadenosine. These methylated species are formed by DNA methyltransferase enzymes (MTases) which catalyze the transfer of an activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to form the above methylated nucleotides within their DNA recognition sequences (Cheng, (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 293-318). DNA methylation is an important biological mechanism that regulates gene expression in vertebrate animals including humans (Bird, (2002) Genes Dev. 16, 6-21), Goll, M. G. & Bestor, T. H. Annu. Rev. Biochem. 74, 481-514 (2005) and serves as a species self-code in bacteria. The AdoMet cofactor is universal for most methylation reactions in living organisms. This biologically and chemically active compound is comprised of a positively charged sulfonium center which joins three peripheral parts: the transferable methyl group, the adenosyl moiety and the homoserine moiety. The adenosyl and homoserine moieties typically serve as anchors which are required for discrete binding and correct orientation of the methyl group in a methyltransferase enzyme. The sulfonium center is thought to activate the methyl group for its transfer onto nucleophilic targets. Some methyltransferases also assist in activation of their target molecules by different mechanisms (Klimasauskas and Lukinavicius (2008) Wiley Encyclopedia of Chemical Biology. DOI: 10.1002/9780470048672.wecb335).
The ability of methyltransferases to catalyze sequence-specific, covalent modifications of biopolymers makes them potential tools for biotechnology. Recently, labeling strategies using three types of designer cofactors for DNA methyltransferases have been presented (Klimasauskas and Weinhold, (2007) Trends Biotechnol. 25, 99-104). One such strategy is based on replacing the methylgroup and the homoserine moiety of the natural cofactor S-adenosyl-L-methionine (AdoMet) by an aziridinyl moiety. These analogs confer methyltransferase-directed nucleophilic opening of the aziridine ring and coupling of the whole cofactor molecule to a target adenine or cytosine residue in DNA. Attachment of a fluorophore via a flexible linker to certain positions of the adenosyl moiety may not interfere with cofactor binding. These cofactors, such as 8-amino[1″-(N″-dansyl)-4″-aminobutyl]-5′-(1-aziridinyl)-5′-deoxyadenosine (Pljevaljcic et al., (2003) J. Am. Chem. Soc. 125, 3486-3492) or 8-amino[1″-(N″-biotinyl)-4″-aminobutyl]-5′-(1-aziridinyl)-5′-deoxyadenosine (Pljevaljcic et al., (2004) Methods Mol. Biol. 283, 145-161) can be used for sequence-specific labeling of biomolecules (Pljevaljcic et al., (2004) Chem Bio Chem 5, 265-269). Aziridine derivatives are also disclosed (WO0003587, publ. 2000) which can be used as cofactor for S-adenosyl-L-methionine-dependent methyltransferases. Labeling is carried out by using AdoMet-dependent MTases, and the adenosyl moiety serves as the molecular anchor for cofactor binding.
The second class is N-mustard analogs of AdoMet such as 5′-(diaminobutyric acid)-N-iodoehtyl-5′-deoxy-8-azido-adenosine or 5′-[(N-iodoethyl)propargylamino]-5′-deoxy-adenosine (Zvag et al., (2006) J. Am. Chem. Soc. 128, 2760-2761). These compounds are structurally and mechanistically similar to the aziridine analogs. They undergo methyltransferase-directed coupling of the whole cofactor molecule to a target adenine or cytosine residue in DNA via its iodoethyl group (the coupling is thought to occur via transient formation and opening of an aziridine ring). These analogs contain the anchoring adenosyl moiety and may contain the homoserine side chain as well. A number of two-step labeling/conjugation methods have been proposed using this approach. For example, U.S. Pat. No. 7,465,544, publ. 2007, discloses reacting groups that are ligatable to the cofactor analogs and can also be used as detectable labels.
The third class of AdoMet analogs contain only replacements of the methyl group with an extended allyl (—CH2CH═CH2) or propargyl (—CH2C≡CH) group bound at the activating sulfonium center. These cofactors are named doubly-activated AdoMet analogs since they bear an activating double or triple bond in beta-position to the transferable carbon unit (Dalhoff et al., (2006) Nat. Chem. Biol. 2, 31-32). The adenosyl and homocysteine moieties are the molecular anchors for cofactor binding, and only part of its molecule (the sulfonium bound activated side chain) is transferred onto a target molecule. These cofactors can be used for methyltransferase-directed derivatization and two-step labeling of plasmid DNA (Lukinavicius et al., (2007) J. Am. Chem. Soc. 129, 2758-2759). These analogs are claimed in WO 2006/108678, publ. 2006, and provide the possibility for transferring smaller linear groups (part of the molecule) onto target biomolecules by methyltransferases, which can be used for labeling of DNA.
However, the labeling strategies that exploit the above cofactor analogs (including the doubly-activated cofactors) bear the following shortcomings:
1) The chemistries of the labeling reactions provide a limited selection with respect to the nature of groups attached to target biomolecules, especially small groups. The minimal transferred unit comprises an entire cofactor molecule in the case of N-adenosylaziridine or N-mustard analogs. For the doubly-activated cofactors, a minimal transferable moiety comprises a 3-carbon linear chain (allyl or propargyl) plus a functional group; however, typically, larger transferable units are used (Klimasauskas and Weinhold, (2007) Trends Biotechnol. 25, 99-104). This limits the applicability of the labeling reactions in such cases when minimal changes to a original biomolecules are required. Furthermore, besides the size limitations, applications of labeled biomolecules may impose certain structural requirements to the attached groups. For example, groups such as hydroxymethyl, 1-hydroxyethyl, 2-chloro-1-hydroxyethyl, 2-hydroxyethylthiomethyl cannot be transferred to cytosine DNA in a sequence-specific manner by any methods known to the prior art. Therefore, labeling methods that are able to attach even shorter moieties or those that expand the existing repertoire of linker/functional group combinations are highly desired.
2) All known types of AdoMet analogs are chemically complex and expensive to obtain (multi-step synthetic procedures including numerous purification steps are required (Pljevaljcic et al., (2003) J. Am. Chem. Soc. 125, 3486-3492; Pljevaljcic et al., (2004) Methods Mol. Biol. 283, 145-161; Lukinavicius et al., (2007) J. Am. Chem. Soc. 129, 2758-2759). The availability of labeling reagents may be thus limited due to their high cost.
3) The N-adenosylaziridine and doubly-activated AdoMet analogs are quite unstable chemically and thus exhibit short half-lifes under physiological conditions (C. Dalhoff, (2005) Dokt. Diss, Aachener Beiträger zur Chemie, Bd. 63; ISBN 3-86130-767-7). This may limit the productive incubation time of a labeling reaction to 1-2 hours. They also need to be stored in special buffers at low temperature (−20° C. to −70° C.). These limitations may be critical in applications, when labeling reagents need to be stored for certain periods of time at ambient temperature or unfrozen (in a refrigerator).
4) All previously known labeling reactions make use of cofactor analogs which form high affinity complexes with directing methyltransferases. Therefore the labeling reactions produce inhibitory products—either tightly bound substrate-cofactor conjugates (with the N-aziridine and N-mustard analogs), or the natural reaction product S-adenosyl-L-homocysteine (with doubly-activated cofactors). These products will remain unproductively bound to the methyltransferase, which may limit enzymatic turnovers of the labeling reaction to a single or just a few turnovers, respectively (Klimasauskas and Weinhold, (2007) Trends Biotechnol. 25, 99-104). This in turn may reduce the efficiency of the reaction and require higher amounts of labeling reagents (cofactor analog and methyltransferase) and extended incubation times.
5) The use of the doubly-activated cofactors with long transferable side chains often is inefficient with wild type methyltransferases due to increased steric bulk of the transferable side chain. One solution to this problem is a steric engineering of the cofactor binding pocket in a directing methyltransferase by site-directed mutagenesis (Lukinavicius et al., (2007) J. Am. Chem. Soc. 129, 2758-2759). However it is not clear if this approach will be successful for other enzymes, since successful engineering examples come only from a single class methyltransferase enzymes. This may limit the applicability of the method, especially its expansion to other classes of AdoMet-dependent methyltransferases as directing enzymes.
Therefore, techniques that (i) permit sequence-specific covalent attachment of short functional groups (C1-C4 chains) onto target biomolecules and (ii) use chemically simple and inexpensive compounds are desired.